Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance

Abstract

SUMO modification regulates diverse cellular processes by targeting hundreds of proteins. However, the limited number of sumoylation enzymes raises the question of how such a large number of substrates are efficiently modified. Specifically, how genome maintenance factors are dynamically sumoylated at DNA replication and repair sites to modulate their functions is poorly understood. Here, we demonstrate a role for the conserved yeast Esc2 protein in this process by acting as a SUMO E2 cofactor. Esc2 is required for genome stability and binds to Holliday junctions and replication fork structures. Our targeted screen found that Esc2 promotes the sumoylation of a Holliday junction dissolution complex and specific replisome proteins. Esc2 does not elicit these effects via stable interactions with substrates or their common SUMO E3. Rather, we show that a SUMO-like domain of Esc2 stimulates sumoylation by exploiting a noncovalent SUMO binding site on the E2 enzyme. This role of Esc2 in sumoylation is required for Holliday junction clearance and genome stability. Our findings thus suggest that Esc2 acts as a SUMO E2 cofactor at distinct DNA structures to promote the sumoylation of specific substrates and genome maintenance.

Keywords: Esc2; SUMO E2; genome maintenance; homologous recombination.

Figure 1.

Esc2 promotes the sumoylation of a specific set of Mms21 substrates. (A) Sumoylation levels of the subunits of the Sgs1-Top3-Rmi1 complex are reduced in esc2Δ cells. Cells containing His8-tagged SUMO were treated with 0.03% MMS for 2 h to induce STR sumoylation. Sumoylated proteins were isolated using Ni-NTA resins and examined by immunoblotting using antibodies recognizing the tag fused to the endogenous Sgs1, Top3, or Rmi1 to visualize sumoylated forms of the proteins (-S), which are indicated by lines next to the blots. Loading is shown by Ponceau S stain (stain). (WT) Wild type. Similar methods for examining sumoylation and annotation of immunoblots are used in subsequent panels unless otherwise noted. (B) esc2Δ reduces the levels of mono-sumoylated form of Pol2 and di-sumoylated form of Mcm3. (Left) HA-tagged Pol2 was immunoprecipitated and its sumoylated form was detected by immunoblotting using anti-SUMO antibody as shown previously (Meng et al. 2019). The unmodified band was detected against the tag (HA) for equal loading control. (Right) Mcm3 sumoylation was examined as in A. (C) esc2Δ cells maintain the sumoylation levels of Yku70 and Rfa1. Yku70 sumoylation was examined as in B (left) and as shown previously (Zhao and Blobel 2005), whereas Rfa1 sumoylation was examined as in A as shown previously (Chung and Zhao 2015). (D) esc2Δ cells maintain the sumoylation levels for the subunits of three SMC complexes, namely cohesin, condensin, and the Smc5/6 complex. Sumoylation of Smc2 was examined as in B (left), and sumoylation of the other proteins was examined as in A. (E) Summary of the effects of esc2Δ on Mms21-dependent sumoylation of DNA metabolism proteins. Results in A–D show that esc2Δ reduces the sumoylation of proteins known to associate with HJ and replication fork structures but not those that mainly interact with ssDNA or dsDNA.

Figure 2.

Examination of Esc2 interactions with Ubc9, SUMO, STR, and the Smc5/6 complex. (A) Smc5 coimmunoprecipitates with Sgs1 but not Esc2. Cells containing endogenously Myc-tagged Esc2 and HA-tagged Sgs1, with or without TAP-tagged Smc5, were examined by co-IP tests. Representative immunoblots examining co-IP eluate (IP) and the whole cell extract (WCE) are shown. (B) Esc2 interacts with Ubc9 and SUMO (Smt3), but not subunits of the Smc5/6 complex and the STR complex nor Pol2 and Mcm3 in yeast-two-hybrid assays. (AD) Gal4 activation domain, (BD) Gal4 DNA binding domain, (vec) vector. SC-Leu-Trp media (-L-T) select for BD and AD plasmids, while SC-Leu-Trp-Ade media (L-T-A) report for positive interactions. (C) Esc2 does not coimmunoprecipitate with Sgs1. Similar levels of Sgs1 were detected in IP samples regardless of whether cells contain Myc-tagged Esc2 or not, reflecting nonspecific binding of Sgs1 to the beads. (D) Smc5 copurifies with Sgs1 regardless of the Esc2 status. Experiments were done as in A. (E,F) Protein binding assays showing that Esc2 binds to Ubc9 but not SUMO or a SUMO chain. Purified GST or GST-Esc2 proteins bound to glutathione beads were examined for their abilities to pull down Ubc9 (E), SUMO (F, lanes 1–6), or SUMO chain composed of four tandem SUMO moieties (F, lanes 7–12). Proteins were examined by SDS-PAGE, and pictures of representative gels after Coomassie blue stain are shown. (S) Supernatant, (W) wash, (E) eluate.

Figure 3.

Esc2 binding to Ubc9 is required for efficient sumoylation of the STR complex, Pol2 and Mcm3. (A) A schematic of Esc2 protein domains. (DNA) DNA-binding domain that prefers to bind HJ and fork structures, (SLD1) SUMO-like domain 1, (SLD2) SUMO-like domain 2. (B) Mutating the SLD2 but not SLD1 domain of Esc2 abolishes its interaction with Ubc9 in yeast two-hybrid assays. Experiments were done and data are presented as in Figure 2B. (C) Esc2-SLD2m abolished Ubc9 interaction in vitro. Experiments were done and data are presented as in Figure 2E. Dotted line denotes removal of superfluous lanes. (D) esc2-SLD2m, but not -SLD1m, reduces sumoylation levels of the STR subunits in cells. Experiments were done and data are presented as in Figure 1A. (E) esc2-SLD2m reduces levels of monosumoylation of Pol2 and disumoylation of Mcm3 in vivo. Experiments were done and data are presented as in Figure 1B.

Figure 4.

Esc2 aids sumoylation via its SLD2 binding to the Ubc9 backside. (A) Mms21-Smc5 stimulates Sgs1 and Top3 sumoylation in the presence of SUMO or SUMO-D68R. In vitro sumoylation assays were performed by incubating purified STR complex with the SUMO E1, the SUMO E2, SUMO (or SUMO-DR), and ATP in the presence or absence of the Mms21-Smc5 SUMO E3 at 30°C for the indicated time (for details, see the Materials and methods). Sgs1 tagged with FLAG and Top3 tagged with CBP and their sumoylated forms were detected by immunoblotting against the tags fused to them. Asterisk indicates a cross-reaction band. (B) Esc2, but not Esc2-SLD2m, stimulates Sgs1 and Top3 sumoylation in vitro. Sumoylation assays were performed as in panel A in the presence of Mms21-Smc5 and SUMO-DR, except Top3 is tagged with V5. The inclusion of Esc2 or Esc2-SLD2m is indicated. (C) Schematics of two Esc2 variants wherein its SLD2 is replaced by SUMO (Esc2-SLD2Δ-Su) or by SUMO-D68R (Esc2-SLD2Δ-SuDR). (D) Esc2-SLD2Δ-Su, but not esc2-SLD2Δ-SuDR, protein interacts with Ubc9 in vitro. GST pull-down tests were performed and results are presented as in Figure 2E. (E) SUMO, but not SUMO-DR, can replace the SLD2 of Esc2 in stimulating Sgs1 and Top3 sumoylation. Sumoylation assays were performed as in A in the presence of Mms21-Smc5 and SUMO-DR, except with shorter time courses. (F) SLD2 can be replaced by SUMO, but not SUMO-DR, to support STR sumoylation in cells. Experiments were done and data are presented as in Figure 1A.

Figure 5.

Esc2 binding to Ubc9 curbs levels of GCR and recombination intermediates. (A) esc2Δ and esc2-SLD2m mutants increase GCR rates. For each genotype, the median rate of at least nine cultures was calculated from two biological duplicates. Error bars are 95% confidence intervals. Two-tailed Mann–Whitney tests were performed for statistical analysis. (B) 2D gel data show that esc2-SLD2m mutants increase X-mol levels at two genomic sites. α-Factor-arrested G1 cells were released into media containing MMS for 2 h. Samples were examined by 2D gel followed by Southern blotting using probes at ARS315 or ARS1212. (Left) A schematic of 2D gel images with X-mol spike indicated by red arrowhead. (Middle) Representative 2D gel images. (Right) Quantification of relative X-mol levels from two different spore clones per genotype, with error bars representing standard deviations. WT level was set to 1.0, and the P-value is derived by Student's t-test. (C) Tetrad analyses from diploid strains with indicated genotype. Spore clones were grown for 2 d at 30°C. Spores containing different mutations are identified based on genotyping. One representative tetrad among at least nine tetrads per diploid strain is shown. (D) esc2-SLD2m worsens genotoxic sensitivity of slx4Δ and mms4Δ cells. Cells were spotted in 10-fold serial dilutions and grown for 2 d at 30°C. (E) A model for Esc2 stimulation of specific substrate sumoylation. Esc2 binding to the backside of Ubc9 (SUMO E2) through its SLD2 leads to the stimulation of sumoylation of a subset of Smc5-6-Mms21 E3 substrates, likely at HJ and replication fork sites, contributing to HJ dissolution and genome stability.